Cell-free protein expression using rolling circle amplification product

ABSTRACT

Methods for in vitro transcription and translation from an RCA product are provided. The methods comprise providing a double-stranded RCA product, wherein the double-stranded RCA product consists essentially of tandem repeats of a minimalistic expression sequence. The methods further comprise expressing a protein from the double-stranded RCA product in a cell-free expression system.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/145,838, filed on May 4, 2016, entitled “Cell-Free Protein ExpressionUsing Rolling Circle Amplification Product”.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 17, 2018, isnamed 285524-3_SL.txt and is 11,160 bytes in size.

FIELD OF INVENTION

The invention generally relates to the generation of a rolling circleamplification product via rolling circle amplification of adeoxyribonucleic acid (DNA) mini-circle having a minimalistic expressionsequence. It further relates to improved cell-free protein expressionsystems that involve in vitro transcription and translation of therolling circle amplification product.

BACKGROUND

Cell-free protein expression provides a simple and efficient method forgenerating proteins without the complications of cell culture, cellengineering, or cell transfection. Cell-free systems for expressingrecombinant proteins address various limitations of cell-basedexpression systems such as protein toxicity, protein degradation,protein aggregation and misfolding, uncontrolled post-translationalmodification, or negative effects of protein expression on cell growthdue to sequestration of cellular machinery. Significantly higherquantities of proteins can be expressed in a shorter period of timeusing a cell-free protein expression system that can be employed fordownstream high-throughput structural and functional analyses. Such invitro protein expression also has significant advantages in terms ofcost savings, streamlined production, easier scale-up, and simplifiedpurification. In a cell-free protein expression system, a desiredprotein of interest is expressed by adding a DNA or RNA that encodes agene of the protein of interest to a transcription-translation-competentcellular extract, and performing the transcription and/or translation ofthe gene of interest. The transcription and translation may be coupledin a single reaction to enable immediate translation of a newlysynthesized mRNA into protein (coupled in vitrotranscription-translation system or coupled transcription-translation ina cell-free system). The coupled in vitro transcription and translationgenerally increases the yield of expressed proteins with less time andin vitro manipulation. The immediate translation of the mRNA avoidspossible adverse effects associated with mRNA degradation or misfolding.

One limitation of in vitro transcription-translation systems is thatthey require large quantities (generally in microgram quantities) of aDNA template. Generally, sufficient amounts of DNA can be obtainedthrough multiple workflow steps and significant labor effort, forexample, by cloning the DNA into a plasmid vector and propagating theplasmid in a host cell (e.g., E. coli) or by synthesizing DNA frommultiple polymerase chain reactions (PCR). However, PCR is often notamenable for large-scale generation of high-quality DNA, due in part tothe high mutation rate of PCR Additionally, the thermal cycling of PCRreactions is difficult to scale-up to larger reactions due tolimitations on how quickly temperatures can be ramped in large volumes.Moreover. PCR products, being linear DNA sequences, may be rapidlydegraded by the action of nucleases that are present in cell-freetranscription-translation extracts. Further, sub-cloning of a gene ofinterest into a plasmid vector followed by high-scale propagation in E.coli through genetic selection is time-consuming and labor intensive.

Isothermal DNA amplification techniques such as rolling circleamplification (RCA) can be employed to generate large quantities ofhigh-quality DNA with less effort, time, and expense, starting from acircular nucleic acid template. Rolling circle amplification reactionsare isothermal, making scale-up to larger reaction sizes straightforwardas there is no requirement for rapid heating and cooling. Rolling circleamplification generates RCA products that are tandem repeat units(concatamers) of the template nucleic acid sequence. RCA of a plasmidDNA, followed by coupled in vitro transcription and translation, ispossible to generate the protein of interest. However, these plasmidsare created via standard cloning methods involving genetic-selectioninside a host cell such as E. coli. Such plasmids therefore contain manyadditional coding and non-coding sequences including sequences for theorigin of replication (for example, oriC), antibiotic selection (forexample, amp for beta-lactamase), and accessory sequences that are usedfor selection and/or screening plasmids in the host cells, such as lacZ,beta-galactosidase. Transcription and/or expression of these ancillarysequences are not desired, or may be considered inefficient, relative tothe gene of interest that is meticulously sub-cloned into the plasmid.Consequently, PCR amplification of a gene of interest within the plasmidis often employed for cell-free protein expression.

There exists a need for improved in vitro transcription and translationsystems for easy generation of desired proteins that are transcribed andtranslated from a DNA that is optimally free of any of the extraneoussequences and host cell contaminants, and does not require PCRsynthesis. Also, it is desirable to increase the yield of cell-freeprotein systems using methods that are simplified and lesstime-consuming.

BRIEF DESCRIPTION

In some embodiments, a method for in vitro transcription and translationusing RCA product is provided. The method comprises the steps of (a)providing a double-stranded rolling circle amplification (RCA) product,wherein the double-stranded RCA product consists essentially of tandemrepeats of a minimalistic expression sequence, and (b) expressing aprotein from the double-stranded RCA product in a cell-free expressionsystem. The minimalistic expression sequence consists essentially of apromoter, an open reading frame, a ribosomal binding site and atranslational termination sequence.

In some embodiments, a method for in vitro transcription and translationusing DNA mini-circle is provided. The method comprises the steps of (a)providing a deoxyribonucleic acid (DNA) mini-circle, wherein the DNAmini-circle consists essentially of a minimalistic expression sequence,(b) generating a double-stranded rolling circle amplification (RCA)product via a rolling circle amplification of the DNA mini-circle, and(c) expressing a protein from the double-stranded RCA product in acell-free expression system. The minimalistic expression sequenceconsists essentially of a promoter, an open reading frame, a ribosomalbinding site and a translational termination sequence.

DRAWINGS

These and other features, aspects and advantages of the invention willbecome better understood when the following detailed description is readwith reference to the accompanying figures.

FIG. 1 illustrates a schematic representation of an embodiment of amethod of in vitro transcription and translation using an RCA productderived from a mini-circle.

FIG. 2 illustrates the yield of an RCA product derived from DNAmini-circles using different primers and reaction conditions.

FIG. 3 illustrates the increased expression of an enhanced greenfluorescent protein (EGFP) when an RCA product derived from a DNAmini-circle was used for in vitro transcription and translation ascompared to PCR-amplified DNA templates, wherein the EGFP coding regionwas designed using contextual codon preference.

FIG. 4 illustrates the enhanced expression of EGFP when an RCA productderived from DNA mini-circle was used for in vitro transcription andtranslation as compared to PCR-amplified DNA templates, wherein the EGFPcoding region was designed using individual codon preference.

FIG. 5 illustrates yields of EGFP expression by in vitro transcriptionand translation using an RCA product derived from a DNA mini-circle,with or without thioated nucleotides, and a PCR-amplified DNA withoutany thioated nucleotides.

FIG. 6 illustrates yields of EGFP expression by in vitro transcriptionand translation using an RCA product derived from a DNA mini-circle,with or without thioated nucleotides, and a PCR-amplified DNA withoutany thioated nucleotides.

FIG. 7 depicts an SDS-PAGE gel illustrating the expression ofBODIPY-labeled human interleukin 2 (IL-2, ˜16 kD) by coupled in vitrotranscription and translation using RCA products derived from DNAmini-circles in comparison to PCR-amplified DNA templates and a notemplate control (NTC) expression reaction.

FIG. 8 illustrates the cell-free expression yield of IL-2 by imagedensitometry of FIG. 7.

FIG. 9 illustrates enhanced expression of EGFP by in vitro transcriptionand translation of an RCA product that is generated from DNAmini-circles having minimalistic expression sequences in comparison withan RCA products derived from a plasmid DNA.

DETAILED DESCRIPTION

The following detailed description is exemplary and not intended tolimit the invention or uses of the invention. Throughout thespecification, exemplification of specific terms should be considered asnon-limiting examples. The singular forms “a”, “an” and “the” includeplural referents unless the context clearly dictates otherwise.Approximating language, as used herein throughout the specification andclaims, may be applied to modify any quantitative representation thatcould permissibly vary without resulting in a change in the basicfunction to which it is related. Accordingly, a value modified by a termsuch as “about” is not to be limited to the precise value specified.Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,so forth used in the specification and claims are to be understood asbeing modified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. Where necessary, ranges have been supplied and thoseranges are inclusive of all sub-ranges there between. To more clearlyand concisely describe and point out the subject matter of the claimedinvention, the following definitions are provided for specific terms,which are used in the following description and the appended claims.

As used herein, the term “nucleoside” refers to a glycosylamine compoundwherein a nucleic acid base (nucleobase) is linked to a sugar moiety. A“nucleotide” refers to a nucleoside phosphate. A nucleotide may berepresented using alphabetical letters (letter designation)corresponding to its nucleoside as described in Table 1. For example, Adenotes adenosine (a nucleoside containing the nucleobase, adenine), Cdenotes cytidine. G denotes guanosine, U denotes uridine, and T denotesthymidine (5-methyl uridine). W denotes either A or T/U, and S denoteseither G or C. N represents a random nucleoside, and dNTP refers todeoxyribonucleoside triphosphate. N may be any of A, C, G, or T/U.

TABLE 1 Letter designations of various nucleotides. Symbol LetterNucleotide represented by the symbol Letter G G A A T T C C U U R G or AY T/U or C M A or C K G or T/U S G or C W A or T/U H A or C or T/U B Gor T/U or C V G or C or A D G or A or T/U N G or A or T/U or C

As used herein, the term “nucleotide analogue” refers to compounds thatare structurally analogous to naturally occurring nucleotides. Thenucleotide analogue may have an altered phosphate backbone, sugarmoiety, nucleobase, or combinations thereof. Nucleotide analogues may bea natural nucleotide, a synthetic nucleotide, a modified nucleotide, ora surrogate replacement moiety (e.g., inosine). Generally, nucleotideanalogues with altered nucleobases confer, among other things, differentbase pairing and base stacking proprieties. As used herein, the term“LNA (Locked Nucleic Acid) nucleotide” refers to a nucleotide analogue,wherein the sugar moiety of the nucleotide contains a bicyclic furanoseunit locked in a ribonucleic acid (RNA)-mimicking sugar conformation.The structural change from a deoxyribonucleotide (or a ribonucleotide)to the LNA nucleotide is limited from a chemical perspective, namely theintroduction of an additional linkage between carbon atoms at the 2′position and 4′ position (e.g., 2′-C, 4′-C-oxymethylene linkage; see,for example, Singh, S. K., et. al., Chem. Comm., 4, 455-456, 1998, orKoshkin, A. A., et. al., Tetrahedron, 54, 3607-3630, 1998)). The 2′ and4′ position of the furanose unit in the LNA nucleotide may be linked byan O-methylene (e.g., oxy-LNA: 2′-O, 4′-C-methylene-β-D-ribofuranosylnucleotide), an S-methylene (thio-LNA), or an NH-methylene moiety(amino-LNA), and the like. Such linkages restrict the conformationalfreedom of the furanose ring. LNA oligonucleotides display enhancedhybridization affinity toward complementary single-stranded RNA, andcomplementary single- or double-stranded DNA. The LNA oligonucleotidesmay induce A-type (RNA-like) duplex conformations. Nucleotide analogueshaving altered phosphate-sugar backbone (e.g., PNA, LNA) often modify,among other things, the chain properties such as secondary structureformation. A star (*) sign preceding a letter designation denotes thatthe nucleotide designated by the letter is a phosphorothioate modifiednucleotide. For example, *N represents a phosphorothioate modifiedrandom nucleotide. A plus (+) sign preceding a letter designationdenotes that the nucleotide designated by the letter is a LNAnucleotide. For example, +A represents an adenosine LNA nucleotide, and+N represents a locked random nucleotide (i.e., a random LNAnucleotide).

As used herein, the term “oligonucleotide” refers to oligomers ofnucleotides. The term “nucleic acid” as used herein refers to polymersof nucleotides. The term “sequence” as used herein refers to anucleotide sequence of an oligonucleotide or a nucleic acid. Throughoutthe specification, whenever an oligonucleotide or nucleic acid isrepresented by a sequence of letters, the nucleotides are in 5′→3′ orderfrom left to right. For example, an oligonucleotide represented by aletter sequence (W)_(x)(N)_(y)(S)_(z), wherein x=2, y=3 and z=1,represents an oligonucleotide sequence WWNNNS, wherein W is the 5′terminal nucleotide and S is the 3′ terminal nucleotide. Theoligonucleotides or nucleic acids may be a DNA, an RNA, or theiranalogues (e.g., phosphorothioate analogue). The oligonucleotides ornucleic acids may also include modified bases and/or backbones (e.g.,modified phosphate linkage or modified sugar moiety). Non-limitingexamples of synthetic backbones that confer stability and/or otheradvantages to the nucleic acids may include phosphorothioate linkages,peptide nucleic acid, locked nucleic acid, xylose nucleic acid, oranalogues thereof.

As used herein, the term “primer” refers to a short linearoligonucleotide that hybridizes to a target nucleic acid sequence (e.g.,a DNA template to be amplified) to prime a nucleic acid synthesisreaction. The primer may be an RNA oligonucleotide, a DNAoligonucleotide, or a chimeric sequence. The primer may contain natural,synthetic, or modified nucleotides. Both the upper and lower limits ofthe length of the primer are empirically determined. The lower limit onprimer length is the minimum length that is required to form a stableduplex upon hybridization with the target nucleic acid under nucleicacid amplification reaction conditions. Very short primers (usually lessthan 3 nucleotides long) do not form thermodynamically stable duplexeswith target nucleic acid under such hybridization conditions. The upperlimit is often determined by the possibility of having a duplexformation in a region other than the pre-determined nucleic acidsequence in the target nucleic acid. Generally, suitable primer lengthsare in the range of about 3 nucleotides long to about 40 nucleotideslong.

As used herein, the term “random primer” refers to a mixture of primersequences, generated by randomizing a nucleotide at any given locationin an oligonucleotide sequence in such a way that the given location mayconsist of any of the possible nucleotides or their analogues (completerandomization). Thus the random primer is a random mixture ofoligonucleotide sequences, consisting of every possible combination ofnucleotides within the sequence. For example, a hexamer random primermay be represented by a sequence NNNNNN or (N)₆. A hexamer random DNAprimer consists of every possible hexamer combinations of 4 DNAnucleotides, A, C, G and T, resulting in a random mixture comprising 4⁶(4,096) unique hexamer DNA oligonucleotide sequences. Random primers maybe effectively used to prime a nucleic acid synthesis reaction when thetarget nucleic acid's sequence is unknown or for performing awhole-genome amplification reaction. Random primers may also beeffective in priming and producing double-stranded rolling circleamplification (RCA) product rather than single-stranded RCA product,depending on the concentration of primer.

As used herein, the term “rolling circle amplification (RCA)” refers toa nucleic acid amplification reaction that amplifies a circular nucleicacid template (e.g., single/double stranded DNA circles) via a rollingcircle mechanism. Rolling circle amplification reaction is initiated bythe hybridization of a primer to a circular, often single-stranded,nucleic acid template. The nucleic acid polymerase then extends theprimer that is hybridized to the circular nucleic acid template bycontinuously progressing around the circular nucleic acid template toreplicate the sequence of the nucleic acid template over and over again(rolling circle mechanism). The rolling circle amplification typicallyproduces concatamers comprising tandem repeat units of the circularnucleic acid template sequence. The rolling circle amplification may bea linear RCA (LRCA), exhibiting linear amplification kinetics (e.g., RCAusing a single, specific primer), or may be an exponential RCA (ERCA)exhibiting exponential amplification kinetics. Rolling circleamplification may also be performed using multiple primers (multiplyprimed rolling circle amplification or MPRCA) leading to hyper-branchedconcatamers. For example, in a double-primed RCA, one primer may becomplementary, as in the linear RCA, to the circular nucleic acidtemplate, whereas the other may be complementary to the tandem repeatunit nucleic acid sequences of the RCA product. Consequently, thedouble-primed RCA may proceed as a chain reaction with exponentialamplification kinetics featuring a cascade in series ofmultiple-hybridization, primer-extension, and strand-displacement eventsinvolving both the primers and both strands. This often generates adiscrete set of concatemeric, double-stranded nucleic acid amplificationproducts. The RCA may be performed in vitro under isothermal conditionsusing a suitable nucleic acid polymerase such as Phi29 DNA polymerase.Suitable polymerases possess strand displacement DNA synthesis ability.

One or more embodiments are directed to methods for expressing a proteinin a cell free expression system (e.g., an in vitro transcription andtranslation system). In one embodiment, the protein is expressed by invitro transcription and translation of an RCA product that is generatedby rolling circle amplification. These in vitro transcription andtranslation reactions yield proteins that are devoid of any intactcells. Generation of such proteins may be desired in a myriad ofapplications including structural and functional proteomics. Thecell-free expression of such proteins may be particularly desirable fortherapeutic applications.

Cell-free expression generally encompasses two modes: (1) mRNA andprotein are made in a single reaction or (2) mRNA is made in a firstreaction and the resulting mRNA product is added to a second, separatetranslation reaction. The RCA product derived from a DNA mini-circle maybe utilized for either modes. (1) or (2). For example, in oneembodiment, the RCA product may be provided to a “coupled in vitrotranscription-translation reaction”, wherein the RCA product DNA isconverted to an mRNA and the mRNA is simultaneously expressed to aprotein in one reaction mixture containing both the ability to produceRNA and protein. In another embodiment, the RCA product may be providedto a “linked transcription-translation reaction”, wherein the RCAproduct DNA is first converted to mRNA and the mRNA is added separatelyto a translation reaction mixture to express a protein.

One or more embodiments of methods for in vitro transcription andtranslation from a double-stranded RCA product are provided. In oneexemplary embodiment, the method includes the steps of providing adouble-stranded RCA product and expressing a protein from thedouble-stranded RCA product in a cell-free expression system. Thedouble-stranded RCA product consists essentially of tandem repeats of aminimalistic expression sequence, wherein the minimalistic expressionsequence consists essentially of a promoter, an open reading frame, aribosomal binding site and a translational termination sequence.

As noted, the double-stranded RCA product consists essentially of tandemrepeats of a minimalistic expression sequence. The minimalisticexpression sequence includes, at the minimum, a promoter, an openreading frame, a ribosomal binding site, and a translational terminationsequence. It may additionally contain sequences that do not materiallyaffect the in vitro transcription and/or translation of the RCA product.For example, it may further include sequences such as a translationalenhancer sequence, an insulator sequence, or a transcriptionaltermination sequence. However, the minimalistic expression sequence andthe resulting double stranded RCA product do not include any additionalsequences that may negatively impact the in vitro transcription andtranslation of the RCA product. For example, the RCA product excludesany extraneous sequences, such as an origin of replication, antibioticselection gene, or any other accessory sequences that are required forcloning, selection, screening and/or replication in a host cell. Thepresence of such extraneous sequences in the RCA product wouldmaterially affect the transcription and/or translation in a cell-freeprotein expression.

The minimalistic expression sequence is a nucleic acid sequencecontaining a particular gene of interest. The minimalistic expressionsequence may also contain minimal genetic elements or sequences that areneeded for expression (for example, a promoter sequence or enhancersequence) of the gene of particular interest. In one or moreembodiments, the minimalistic expression sequence consists essentiallyof a promoter, an open reading frame, a ribosomal binding site and atranslational termination sequence. Numerous examples of suitablepromoters are known in the art, including, for example, T7 RNApolymerase promoter sequences. Likewise, numerous examples of suitableribosomal binding sites are known in the art, including for examplesinternal ribosome entry sites (IRES), polyA tracts, species-independenttranslational leaders (SITS), Kozak consensus sequences, andShine-Dalgarno sequences. As noted above, the minimalistic expressionsequence may additionally contain elements that do not materially affectthe in vitro transcription and translation of the RCA product. Forexample, in some embodiments, the minimalistic expression sequence mayadditionally contain a translational enhancer sequence, an insulatorsequence, a transcriptional termination sequence, or combinationsthereof. In one embodiment of the method, the minimalistic expressionsequence consists essentially of a promoter, an open reading frame, aribosomal binding site, a translational termination sequence, and aninsulator sequence. The insulator sequence generally enhances theefficiency of ribosomal binding or translational initiation. Numerousexamples of suitable insulator sequences exist in the art, including forexample, sequences encoding poly-histidine tracts. In some embodimentsthe insulator sequence may be determined empirically by inserting spacersequences around the ribosomal binding site or by optimizing orinserting codons within the N-terminus of the expressed protein. Theminimalistic expression sequence may further include a pre-promotersequence, a sequence for protease cleavage or nucleotide cleavage, asequence for protein purification, or combinations thereof. Theminimalistic expression sequence is selected such that it does notcontain any sequences that hampers or inhibits either the transcriptionand/or translation of the desired protein product or otherwise make theprotein production more cumbersome.

In one or more embodiments, the RCA product is generated from a DNAmini-circle as a template, wherein the DNA mini-circle consistsessentially of a minimalistic expression sequence. The RCA product maybe a linear or a branched concatamer, wherein the concatamer containstandem repeats of the minimalistic expression sequence derived from theDNA mini-circle. In a preferred embodiment, the RCA linear concatamer isdouble-stranded. As noted, the DNA mini-circle consists essentially of aminimalistic expression sequence, which means that the DNA mini-circleincludes only minimalistic expression sequence and excludes any sequenceother than the minimalistic expression sequence, such as any extraneoussequences. Thus, amplification of the DNA mini-circle comprising aminimalistic expression sequence may only be accomplished outside of acell.

The “extraneous sequences” includes the sequences which are notnecessary for coding or expression of a desired protein. The extraneoussequences may include the accessory sequences that are used forselection, screening, and/or propagation of a plasmid in a host cell,such as lacZ, beta-galactosidase. The extraneous sequences may includesequences for origin of replication, antibiotic selection gene, suitablerestriction sites for insertion of a gene, such as multiple cloningsites, or combinations thereof. The extraneous sequence may furthercomprise any other sequence required for cloning into a host cell ordetection in a host cell.

In one or more embodiments, the open reading frame of the minimalisticexpression sequence comprises a codon-optimized sequence, a purificationtag sequence, a protease cleavage site or combinations thereof. Togenerate a codon optimized sequence, codon bias, contextual codonpreference, and/or individual codon preference are the factors which aregenerally considered.

The codon-optimized sequence of the open reading frame may enhance therate or quality of translation of the RCA product. Codon optimizationgenerally improves the protein expression by increasing thetranslational efficiency of a gene of interest. The functionality of agene may also be increased by optimizing codon usage within the customdesigned gene. In codon optimization embodiments, a codon of lowfrequency in a species may be replaced by a codon with high frequency,for example, a codon UUA of low frequency may be replaced by a codon CUGof high frequency for leucine. Codon optimization may increase mRNAstability and therefore modify the rate of protein translation orprotein folding. Further, codon optimization may customizetranscriptional and translational control, modify ribosome bindingsites, or stabilize mRNA degradation sites.

In one example, the expression of unexpected high-molecular weightproteins (26 kD-72 kD) was observed after codon-optimization of an RCAproduct 54 (derived from a DNA mini-circle of SEQ ID No. 8 encodinghuman IL-2, having a transcription termination sequence andcodon-optimized for each individual codon) (FIG. 7). In the exampleprovided in FIG. 7, the minimalistic IL2 expression sequences of the RCAproduct or PCR-amplified control DNA (derived from SEQ. ID. No. 7 andSEQ ID No. 8) were virtually identical except for codon usage within theIL-2 open reading frame. The SEQ ID No.8 was codon-optimized accordingto the JCat tool [Grote et al., Nucleic Acids Res. Jul. 1, 2005; 33 (WebServer issue: W 526-531). JCat: a novel tool to adapt codon usage of atarget gene to its potential expression host.], which maximizesindividual codon usage in a target gene to the codon preferences of anexpression host. In contrast, SEQ ID No. 7 was contextually adaptedbased on a codon optimization process starting from the natural codingsequence of human IL-2, wherein only specific sites were re-coded. Thecoding sequence of IL-2 comprises two di-lysine repeats, which werere-coded into polyA tracts by the JCat tool because the AAA codon issignificantly preferred over AAG in E. coli. These di-lysine repeatswere not substantially re-coded in SEQ ID No. 7 through the contextualcodon optimization process. AAAAAA tracts are often known as ribosomalslippery sequences that can frameshift the translated product (Yan etal., Cell. 160:870-81, 2015) and exert additional translational controlthrough ribosomal stalling (Arthur et al., Sci Adv. 1: e1500154, 2015).For linear DNA templates encoding IL2, such a frameshift may alter thetranslated product such that downstream translational stop codons areread-through. However, because the linear DNA template is finite, thecorresponding mRNA transcript is also finite and the ribosome eventuallyreaches the end of the message (after frameshift) and stalls withoutreleasing a translated product. In the example, an RCA product derivedfrom a DNA mini-circle generated from SEQ ID No. 8, an RCA product whichis a concatamer of tandem IL2 repeat sequences comprising AAAAAAribosomal slippery sequences. When polycistronic run-off products aretranscribed from the RCA product by read-through of transcriptionterminator sequences, then ribosomal slippery sequences in thecorresponding polycistronic message contribute to additionalread-through of translational stop codons. Consequently, thepolycistronic message generates high-molecular weight translatedproducts that are not IL-2 but rather off-target, high-molecular weight,and undesirable. In fact, transcriptional termination by RNA polymerasesis highly inefficient due to various factors, includingsequence-specific parameters and environmental parameters that affectRNA folding. Generally T7 RNA polymerase terminates with only 52%efficiency at Class I/II transcriptional termination sequences derivedfrom the E. coli rrnB operon, and the efficiency of transcriptiontermination declines as a function of increasing concentrations of dNTP.Thus the data presented in FIGS. 8 and 9 show that tandem repeats ofcistronic or polycistronic mRNAs are widely generated from an RCAproduct template in coupled transcription-translation reactions (despitethe presence of transcription termination sequences) and thecorresponding mRNA may be effectively or ineffectively processed byribosomes depending in part on codon usage, which ultimately enhances orreduces cell-free protein yield depending on whether the downstreamcistrons in the message are designed appropriately.

In another example of codon optimization, SEQ ID No.2 and SEQ ID No.4includes EGFP open reading frames that are codon-optimized according tothe JCat tool, which maximizes individual codon usage of a target geneto the codon preferences of an expression host. Further, SEQ ID No. 1and SEQ ID No.3 includes open reading frames for EGFP that arecontextually adapted based on the following process; starting from thenatural coding sequence of EGFP, only specific sites were re-coded toavoid cryptic start sites (ATG), cryptic ribosomal binding sites (forexample, AGGA, GAGG, GGAG), class II termination sequences [(A, C, orT)ATCTGTT], ribosomal slippery sequences [NNNYYY, where Y(A, T)], andribosomal pause sites (for example, AGG, GGA, GAG, GGG, GGT, GTG)upstream of internal ATG methionines. The data presented in FIGS. 3 and4 illustrate that the cell-free expression yield of EGFP is partiallyinfluenced by codon usage.

In some embodiments, the open reading frame of the minimalisticexpression sequence comprises a tag sequence for purification of theexpressed protein. The tag sequence may be an affinity tag, tag forprotease cleavage or combinations thereof. The affinity tag may be usedfor rapid purification and detection of recombinant proteins. Theaffinity tag may include a polyhistidine tag (his6 (SEQ ID NO: 9)),Glutathione S-transferase tag (GST), haemagglutinin (HA), myc (derivedfrom c-myc gene product), FLAG (consisting of eight amino acidsAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 10) including anenterokinase-cleavage site) or combinations thereof. Although fusiontags help in rapid purification or detection of the desired protein, thetags may not be considered to be permanent fixtures or domains of therecombinant proteins. Hence, removal of the fusion tag is often neededfor highly analytical studies of recombinant protein structure andfunction. The tag for purification may be removed from the protein byusing another type of tag, such as protease cleavage tag. The proteasecleavage tag may be used to cleave a distinct peptide bond within aspecific protein or peptide sequence. The protease cleavage tag mayinclude, for example, PreScission Protease tag (GE Healthcare) orthrombin protease tag (GE Healthcare).

As noted, in some embodiments, the minimalistic expression sequencefurther consists essentially of a transcription termination sequence.The transcription termination sequence is generally situated at the 3′end of a gene in a DNA template. Transcription termination sequencesprovide signals in the newly synthesized mRNA to initiate the process ofreleasing the mRNA from the transcriptional complex, which can also aidin effective translation of the desired protein product.

The effects of inefficient transcription termination in an RCA productderived from a mini-circle are largely inconsequential compared to anRCA product derived from a plasmid DNA. In some cases, plasmid DNAcontaining a gene of interest must be digested using a restrictionenzyme to create a double-stranded DNA break immediately after the geneto prevent transcription from proceeding beyond that point when RCAproduct is derived from the plasmid. If run-off transcription were tooccur, the other sections of the plasmid containing many coding andnon-coding sequences (including sequences for the origin of replication,antibiotic selection, and accessory sequences that are used forselection, screening and/or propagation of the plasmid in a host cell)would be transcribed. RCA product derived from the plasmid DNA, whenused in an undigested state, may produce unwanted mRNA species, viatranscriptional read-through, that risk production of proteincontaminants together with (or in a greater amount than) the protein ofinterest. However, poor transcription termination in an RCA productderived from a mini-circle may still generate on-target mRNA.Consequently, the yield of cell-free protein is better from an RCAproduct derived from a DNA mini-circle compared to either an RCA productderived from a plasmid or PCR-amplified plasmid DNA. Similar expressionbenefits are observed even when the RCA product derived from a DNAmini-circle is completely devoid of transcription termination sequences,which is an unexpected result. This is illustrated in differentexamples, such as Examples 2 and 3, and the results are depicted inFIGS. 3, 4 and 6. For the examples provided in FIGS. 3 and 4, SEQ IDNo.1 and SEQ ID No.2 encode EGFP with transcription terminationsequences, whereas SEQ ID No.3 and SEQ ID No.4 encode EGFP withouttranscription termination sequences. An RCA product 22 (derived from DNAmini-circles of SEQ ID No.3) generated higher expression of EGFPcompared to a PCR-amplified DNA product 26 (derived from SEQ ID No.3),and overall EGFP yield from RCA product 22 (derived from DNAmini-circles of SEQ ID No.3) was comparable to the PCR-amplified DNAproduct 24 (derived from SEQ ID No. 1) (FIG. 3). Using a different codonoptimization strategy for EGFP, the RCA product 30 (derived from DNAmini-circles of SEQ ID No.4) generated higher amounts of EGFP thaneither the PCR-amplified DNA product 32 (derived from DNA of SEQ ID No.2) or PCR-amplified DNA product 34 (derived from DNA of SEQ ID No.4)(FIG. 4). However, EGFP expression yield was maximal using RCA products20 (derived from DNA mini-circles of SEQ ID No. 1) and 28 (derived fromDNA mini-circles of SEQ ID No. 2). These examples show thattranscriptional termination sequences, while essential for proteinexpression from PCR-amplified DNA, are not essential for robust proteinexpression from RCA products derived from DNA mini-circles. These RCAproducts improve cell-free protein expression by generating tandemrepeats of cistronic mRNA species, wherein every cistron of the mRNAcomprises the desired target gene. The tandem repeats of the cistron mayin turn improve the mRNA stability, particularly when transcriptiontermination signals are absent, and contribute to higher translationalflux of the desired protein product.

In one or more embodiments, the method for in vitro transcription andtranslation employs a double-stranded RCA product as DNA template. Inthese embodiments, the intramolecular ligation of a double-stranded DNAtemplate generates a double-stranded DNA mini-circle, which is employedas a template for the RCA reaction. The RNA polymerases used incell-free transcription reactions (for example, T7 RNA polymerase)generally require double-stranded DNA promoter sequences for effectivebinding to DNA coding sequences. The effective binding of RNA polymeraseto the double-stranded DNA promoter sequence initiates efficienttranscription. Thus, RCA reaction conditions that promote the generationof double-stranded RCA products are desired for effective in vitrotranscription and translation.

In some embodiments, the double-stranded RCA product is provided to thecell-free expression system without any further processing. In oneembodiment, the RCA product is added to the cell-free system directlyafter amplification. The term “further processing” is meant to includean act of restriction digestion, ligation, or combinations thereof ofthe RCA product. However, in some embodiments, the RCA product may beseparated (e.g., by precipitation) to remove salts or any othercontaminants, such as primers or smaller fragmented DNA from thereaction medium before proceeding for cell-free expression using aeukaryotic cell-extract.

In one exemplary embodiment, the double-stranded RCA product has tandemrepeats of a minimalistic expression sequence. In this embodiment, theminimalistic expression sequence is devoid of any extraneous sequencesthat are required for propagation of the DNA in a host cell. Further,the double-stranded RCA products of this embodiment are provided to thecell-free expression system without any restriction digestion and/orligation.

Protein yields from cell-free expression reactions employing RCAproducts generated from DNA mini-circles are much higher than that ofRCA products generated from plasmid DNA, which is described in detail inExample 5 and depicted in FIG. 9. In this example, cell-free yield ofEGFP was enhanced when an RCA product 70 (derived from DNA mini-circlesof SEQ ID No.1 encoding EGFP with a transcription termination sequence)was expressed compared to RCA product 72 (derived from plasmidcomprising SEQ ID No.1). Further, in the absence of a transcriptiontermination sequence, the expression of EGFP was also enhanced using anRCA product 74 (derived from DNA mini-circles of SEQ ID No.3) relativeto RCA product 76 (derived from plasmid DNA comprising SEQ ID No.3).These examples show that the presence of extraneous sequences (such asorigin of replication, antibiotic selection and lacZ selectionsequences) repeated as concatemers within the RCA product of plasmid DNAhinder cell-free expression of the desired protein target.

Cell-free expression of RCA products derived from DNA mini-circles alsoresults in higher protein yield compared to nucleic acids amplified byPCR. For example, synthesis of EGFP protein by coupled in vitrotranscription and translation were compared between PCR-amplified DNAand RCA products derived from DNA mini-circles. The enhanced yield ofEGFP when RCA products 20, 22 and 28, 30 were expressed compared tousing PCR-amplified DNA 24, 26 and 32, 34, respectively, are shown inFIGS. 3 and 4.

In some embodiments, the double-stranded RCA product that is used for invitro transcription-translation reaction comprises thioated nucleotides.In these embodiments, the RCA reactions are supplemented with thioateddNTPs, such as α-S-dATP or α-S-dTTP, into the dNTP mixture for randomincorporation of thioated bases into the RCA DNA product whileamplification. Cell-free protein expression is improved when an RCAproduct comprising thioated nucleotides is used for in vitrotranscription and translation when compared to non-thioated RCAproducts. For example, EGFP protein yield was higher when thioated RCAproduct 36 (derived from a DNA mini-circle of SEQ ID No.5) was expressedrelative to either PCR-amplified DNA product 42 (derived from DNA of SEQID No.5) or non-thioated RCA product 38 (derived from a DNA mini-circleof SEQ ID No.5 with AT hexamers) or non-thioated RCA product 40 (derivedfrom a DNA mini-circle of SEQ ID No.5 with random hexamer), as shown inFIG. 5. Further, in another example wherein transcription terminationsequences were removed downstream of the EGFP coding sequence, cell-freeEGFP protein yield was much higher when thioated RCA product 44 (derivedfrom DNA mini-circles of SEQ ID No.6) was expressed compared to eithernon-thioated PCR-amplified DNA product 48 (derived from DNA of SEQ IDNo.6) or non-thioated RCA product 46 (derived from a DNA mini-circle ofSEQ ID No.6 with AT hexamer), as shown in FIG. 6.

The present method for cell-free protein expression includes in vitrotranscription and in vitro translation. The in vitro transcriptionreaction employs double-stranded RCA product, wherein thedouble-stranded RCA product consists essentially of tandem repeats of aminimalistic expression sequence. The minimalistic expression sequenceincludes a promoter sequence. The promoter sequence is present upstream(5′) of the gene of interest to be transcribed. DNA-dependent RNApolymerases bind to the double-stranded DNA promoter region to initiategene transcription. A variety of suitable RNA polymerases is known inthe art and includes those having only one subunit (for example, thosefrom bacteriophages like T3 and T7, and mitochondria) as well asmulti-domain RNA polymerases derived from bacteria and eukaryotes. TheRNA polymerase may further require additional protein co-factors forefficient transcription.

In some embodiments of the cell-free translation reaction, abiomolecular translational machinery is extracted from cells andutilized for in vitro translation. In one or more embodiments, the codonoptimized sequence of the open reading frame enhances the rate oftranslation. The composition, proportion of enzymes, and building blocksrequired for translation are provided by the cell-free extract or may besupplemented with synthetic components. The mRNAs synthesized bytranscription are expressed in a translation reaction, which producesthe target protein in the cell-free extract. In some embodiments of thein vitro expression reaction, protein synthesis occurs in cell-freeextract rather than within cultured cells (The extracted material fromcells may be referred to herein as a “cell-free extract” or “cellextract”). The cell extract contains generally the cytosolic andorganelle components of the cell. The cell-free extract may supply allor most of the molecules required for cell-free transcription andtranslation, such as ribosomes for translation, tRNA and amino acids,enzymatic cofactors and an energy source, and cellular componentsessential for protein folding.

In some embodiments, the cell-free expression system comprises aprokaryotic cell extract, a eukaryotic cell extract, or a combinationthereof. In yet another embodiment, the cell-free expression system isformulated from individually-purified components. In one embodiment, thecell extract developed for cell-free protein expression is derived fromprokaryotic organisms. In this embodiment, the mRNA derived from RCAproduct DNA may be added to, or produced within, the prokaryotic extractto express a protein. The prokaryotic extracts capable of supportingtranslation may be derived from E. coli. In some other embodiments, thecell extract is derived from eukaryotic cells, such as protozoans, yeastcells, insect cells, mammalian cells, or human cells. In theseembodiments, the mRNA derived from RCA product DNA may be added to, orproduced within, the eukaryotic cell extract, such as, rabbitreticulocvte lysates (RRL), wheat germ extracts, insect cell lysates(such as SF9 or SF21), mammalian lysates (such as CHO), human lysates(such as HeLa), or protozoan lysate (such as Leishmania). The cell-freeextracts derived from eukaryotic systems contain the necessary cellularmacromolecules, such as ribosomes, translation factors and tRNAsrequired for efficient protein synthesis, wherein energy sources andamino acids may need to be supplemented.

In one exemplary embodiment, the nucleic acid template for RCA reactionis a deoxyribonucleic acid (DNA) template. The DNA template may be asynthetic DNA or a natural DNA. The DNA template may be a circular DNAtemplate, a linear DNA template, or a nicked DNA template. In someembodiments, the nucleic acid template is a DNA mini-circle template,and RCA is used to amplify the DNA mini-circle template in a cell-freesystem. In one example embodiment, the circularization of the linearnucleic acid template is accomplished by an enzymatic reaction, forexample, by incubation with a ligation enzyme such as DNA ligase. Insome embodiments, the DNA mini-circle template includes a minimalisticexpression sequence. In some embodiments, the RCA of the DNA mini-circletemplate contains a minimalistic expression sequence to produce a tandemrepeat DNA sequence. The produced tandem repeat sequence consistsessentially of multiple units of the minimalistic expression sequence.The RCA product used for in vitro transcription-translation may be in anintact, non-degraded state.

The rolling-circle amplification reaction often employs reagents such asa primer, a polymerase, and free nucleotides (dNTPs). In someembodiments. RCA may be performed by contacting a double-stranded DNAmini-circle with a primer solution comprising a random primer mixture toform a nucleic acid template-primer complex; contacting the nucleic acidtemplate-primer complex with a DNA polymerase and deoxyribonucleosidetriphosphates; and amplifying the nucleic acid template. The nucleicacid polymerase that is employed in the amplification reaction may be aproofreading nucleic acid polymerase. RCA may be performed by using anyof the DNA polymerases that are known in the art, including, but notlimited to, a Phi29 DNA polymerase. The amplification reaction mixturemay further include additional reagents such as suitable amplificationreaction buffers.

In some embodiments, each of the reagents used in the nucleic acidamplification reaction may be pre-treated to remove any contaminatingnucleic acids. In some embodiments, the pre-treatment of the reagentsincludes incubating the reagents in presence of ultraviolet radiation.In some other embodiments, the reagents are de-contaminated byincubating the reagents in presence of a nuclease and its co-factor (forexample, a metal ion). Suitable nucleases include, but are not limitedto, exonucleases such as exonuclease I or exonuclease III. In someembodiments, the proofreading DNA polymerases used for DNA amplificationreaction may be de-contaminated by incubating with a divalent metal ion(for example, magnesium or manganese ions) in absence of dNTPs.

The RCA reaction may be performed using a random primer mixture. In someembodiments, specific primers are used for the RCA reaction. Primersequences comprising one or more nucleotide analogues may also be used.In one or more embodiments, the RCA is performed using a random primermixture comprising a nucleotide analogue. The nucleotide analogue may bean inosine, a Locked Nucleic Acid (LNA) nucleotide, a Peptide NucleicAcid (PNA) nucleotide, a thioated nucleotide, 2-amino-deoxyadenosine,2-thio-deoxythymidine, a polycation nucleotide, Zip Nucleic Acid (ZNA)polycation modified nucleotide, or combinations thereof. In one or moreembodiments, the random primer mixture has a sequence+N+N(atN)(atN)(atN)*N (AT hexamer Primer). In some embodiments,nuclease-resistant primers (e.g., primer sequences comprisingphosphorothioate groups at appropriate positions) are employed for theamplification reactions (e.g., NNNN*N*N). In some embodiments, theamplification of the DNA mini-circles employs random hexamers or ahexamer primer, +N+N(at N)(at N)(at N)*N (AT hexamer primer), where “N”represents a random nucleotide (i.e., N may be any of A, C, G, or T/U),“at N” represents a random mixture containing 2-amino dA, 2-thio-dT,normal G and normal C, a plus (+) sign preceding a letter designationdenotes that the nucleotide designated by the letter is a locked nucleicacid (LNA) nucleotide, a star (*) sign preceding a letter denotes thatthe nucleotide designated by the letter is a phosphorothioate modifiednucleotide.

During the amplification reaction, the DNA mini-circle template isreplicated by a polymerase in the presence of deoxyribonucleosidetriphosphates (dNTPs) or their modified counterparts. The freenucleotides employed in nucleic acid template amplification may includenatural nucleotides (for example, dATP, dGTP, dCTP or dTTP) or theirmodified analogues. In some embodiments, the reaction mixture issupplemented with thioated dNTPs. The thioated dNTPs may include but arenot limited to α-S-dGTP, α-S-dCTP, α-S-dATP, and α-S-dTTP. The thioateddNTPs such as α-S-dATP or α-S-dTTP may be added into the dNTP mixturefor random incorporation of the thioated bases into the RCA product.

In some embodiments, the RCA is performed using a final concentration ofdNTPs in a range of about 10 μM to about 10 mM. In one or moreembodiments of RCA reactions, the dNTP concentration is less than 10 mM.In these embodiments, the concentration of dNTPs is kept lower than 10mM to avoid hydrogel formation from the RCA product and to remain at aconcentration below or equal to the amount of divalent cation (e.g.magnesium) present in the reaction buffer. Hydrogel formation may occurafter amplification in the presence of a high concentration of dNTPswhich may further complicate the downstream manipulation such aspipetting and processing of the RCA product. Hydrogel formation may beobserved when dNTP concentration of 50 mM or more is used in the RCAreaction.

RCA may be performed using commercially available RCA amplification kitssuch as Illustra™ TempliPhi™ Amplification Kit (GE Healthcare).TempliPhi rolling-circle amplification employs modified random primers,which provide higher sensitivity and amplification balance. In someembodiments, nuclease-resistant primers are used for RCA reaction. Sincehigh concentration of template DNA is required for the present method ofin vitro transcription and translation, a more balanced DNAamplification with faster kinetics and higher yield may be achievedusing RCA.

A variety of methods may be used to prepare a DNA mini-circle templatefor use with methods of the invention. In some embodiments, a linear DNAtemplate may be circularized to generate a DNA mini-circle template. Inone example embodiment, the circularization of the linear DNA templatemay be effected by an enzymatic reaction, for example, by incubationwith a ligation enzyme such as DNA ligase. In some embodiments, theterminal ends of the linear DNA template are hybridized to a nucleicacid sequence such that the terminal ends come in close proximity.Incubating with a ligation enzyme may then effect the circularization ofthe hybridized linear DNA template to generate a DNA mini-circle.Suitable DNA mini-circle template may also be generated by PCRamplification of a portion of a larger DNA (for example, a genomic DNA,or a DNA from a DNA library) using appropriate PCR primers, followed bycircularization of the PCR product. DNA mini-circle may also begenerated by chemical synthesis of suitable linear oligonucleotidesfollowed by circularization of the synthesized oligonucleotide. In someembodiments, the synthesized linear oligonucleotides may consistessentially of minimalistic expression sequence and achievecircularization via DNA ligase to generate DNA mini-circle.

One or more of the methods may further comprise steps of purifying,analyzing and/or quantifying the DNA mini-circles. Isolation orpurification of the dsDNA mini-circles and/or removal of thecontaminants, such as enzymes or non-ligated form of DNA may beperformed prior to the amplification reaction. Any suitable techniquesthat are used for purification, analysis or quantification of nucleicacids may be employed. Non-limiting examples include precipitation,filtration, affinity capture, gel electrophoresis, sequencing or HPLCanalysis. For example, the purification of the circular nucleic acidsmay be achieved by affinity capture. In some embodiments, the methodsmay further comprise processing of the generated DNA mini-circle.Post-processing of the generated DNA mini-circle may vary according tothe intended use.

EXAMPLES

Unless specified otherwise, ingredients described in the examples arecommercially available from common chemical suppliers. Someabbreviations used in the examples section are expanded as follows:“mg”: milligrams; “ng”: nanograms; “pg”: picograms; “fg”: femtograms:“mL”: milliliters: “mg/mL”: milligrams per milliliter; “mM”: millimolar:“mmol”: millimoles; “pM”: picomolar; “pmol”: picomoles; “μL”:microliters: “min.”: minutes and “h.”: hours.

FIG. 1 is a schematic representation of one of the general embodimentsof the methods of in vitro transcription and translation using an RCAproduct derived from a DNA mini-circle. A DNA expression construct 10that consists essentially of a minimalistic expression sequence 12 iscircularized by a ligation reaction to generate a DNA mini-circle 14.The DNA mini-circle 14 is amplified by RCA. The amplification reactiongenerates a concatamer RCA product 16 having tandem repeat units of theexpression construct 10, containing the minimalistic expression sequence12, which is devoid of any extraneous sequences. As such, the DNAmini-circle is not amenable to propagation inside a cell. The RCAproduct 16 is then expressed in an in vitro transcription-translationreaction. In some examples, the minimalistic expression sequence of theDNA mini-circle 14 contains a transcription termination sequence. TheRCA product formed from such a DNA mini-circle will have a transcriptiontermination sequence in each of the tandem repeats. Subsequently, themRNA 17 produced from the RCA product 16 of this example may beterminated at the transcription termination sequence at each tandemrepeat units. However, if read-through of any one of the transcriptiontermination sequences occurs (such as the mRNA 18), the downstream mRNAsequence still encodes for the anticipated protein target. In some otherexamples, the minimalistic expression sequence of the DNA expressionconstruct 10 does not contain a transcription termination sequence. ThemRNA 18 produced from the RCA product of such examples does not getterminated at each tandem repeat unit, and thus transcribes continuouslythrough multiple tandem repeat sequences of the RCA product. However, ineither case, the mRNA is translated into a protein with a desiredsequence 19 due to the presence of translational termination sequencesin the DNA expression construct 10.

Example 1: Rolling Circle Amplification of DNA Mini-Circle

Generation of DNA Mini-Circle:

A minimalistic expression sequence for EGFP and human interleukin-2(IL-2) were designed in silico and synthesized in vitro. All of theseexpression sequences contained a T7 promoter and +1 sequence (firstribobase position of the 5′ untranslated region of the resulting mRNA)followed by a T7 phi10 promoter stem-loop sequence. A variety ofadditional non-coding and coding parameters were included during thedesigning of the minimalistic expression sequence, including; T7pre-promoter sequences, ribosomal binding sequences, insulator sequencesfor enhancing ribosomal binding, insulator sequences for enhancingribosomal initiation, transcription termination sequences, translationalstart and stop sequences, leader-sequence protease cleavage sites, andcodon optimization. Representative minimalistic expression sequenceswith these parameters are listed as SEQ ID No. 1-8 (Table 2). Thesequences range in size from 655-970 base pairs.

TABLE 2  Sequences of various minimalistic expression sequences. SEQID No. Sequences 1 CCGGAATTCGGATCCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGCGTAAGGAGGTTTGGAATGCATCACCATCACCATCACGGCTCACTGGAAGTTCTGTTCCAGGGGCCCGGCTCAGTAAGCAAGGGCGAAGAGCTGTTCACCGGGGTTGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAAGGCGAAGGCGACGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAATCCGCCATGCCCGAAGGCTACGTCCAAGAGCGCACCATCTTCTTCAAAGACGACGGCAACTACAAGACCCGCGCCGAAGTGTGTTCGAAGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAAGAAGACGGCAACATCCTGGGGCACAAGCTCGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAAGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAAAAGCGCGATCACATGGTCCTGCTCGAGTTCGTGACCGCCGCCGGCATCACTCTCGGCATGGACGAGCTGTACAAGTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGCGCGGATCCGGTAC CCCG 2CCGGAATTCGGATCCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGCGTAAGGAGGTTTGGAATGCATCATCACCATCACCACGGCTCACTGGAAGTTCTGTTCCAGGGGCCCGGCTCAGTTTCTAAAGGTGAAGAACTGTTCACCGGTGTTGTTCCGATCCTGGTTGAACTGGACGGTGACGTTAACGGTCACAAATTCTCTGTTTCTGGTGAAGGTGAAGGTGACGCTACCTACGGTAAACTGACCCTGAAATTCATCTGCACCACCGGTAAACTGCCGGTTCCGTGGCCGACCCTGGTTACCACCCTGACCTACGGTGTTCAGTGCTTCTCTCGTTACCCGGACCACATGAAACAGCACGACTTCTTCAAATCTGCTATGCCGGAAGGTTACGTTCAGGAACGTACCATCTTCTTCAAAGACGACGGTAACTACAAAACCCGTGCTGAAGTTAAATTCGAAGGTGACACCCTGGTTAACCGTATCGAACTGAAAGGTATCGACTTCAAAGAAGACGGTAACATCCTGGGTCACAAACTGGAATACAACTACAACTCTCACAACGTTTACATCATGGCTGACAAACAGAAAAACGGTATCAAAGTTAACTTCAAAATCCGTCACAACATCGAAGACGGTTCTGTTCAGCTGGCTGACCACTACCAGCAGAACACCCCGATCGGTGACGGTCCGGTTCTGCTGCCGGACAACCACTACCTGTCTACCCAGTCTGCTCTGTCTAAAGACCCGAACGAAAAACGTGACCACATGGTTCTGCTGGAATTTGTTACCGCTGCTGGTATCACCCTGGGTATGGACGAACTGTACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGCGCGGATCCGGTACCCCG 3CCGGAATTCGGATCCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGCGTAAGGAGGTTTGGAATGCATCACCATCACCATCACGGCTCACTGGAAGTTCTGTTCCAGGGGCCCGGCTCAGTAAGCAAGGGCGAAGAGCTGTTCACCGGGGTTGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAAGGCGAAGGCGACGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAATCCGCCATGCCCGAAGGCTACGTCCAAGAGCGCACCATCTTCTTCAAAGACGACGGCAACTACAAGACCCGCGCCGAAGTGAAGTTCGAAGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAAGAAGACGGCAACATCCTGGGGCACAAGCTCGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAAGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAAAAGCGCGATCACATGGTCCTGCTCGAGTTCGTGACCGCCGCCGGCATCACTCTCGGCATGGACGAGCTGTACAAGTAATAATACTAGAGCGCGGATCCGGTACCCCG 4CCGGAATTCGGATCCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGCGTAAGGAGGTTTGGAATGCATCATCACCATCACCACGGCTCACTGGAAGTTCTGTTCCAGGGGCCCGGCTCAGTTTCTAAAGGTGAAGAACTGTTCACCGGTGTTGTTCCGATCCTGGTTGAACTGGACGGTGACGTTAACGGTCACAAATTCTCTGTTTCTGGTGAAGGTGAAGGTGACGCTACCTACGGTAAACTGACCCTGAAATTCATCTGCACCACCGGTAAACTGCCGGTTCCGTGGCCGACCCTGGTTACCACCCTGACCTACGGTGTTCAGTGCTTCTCTCGTTACCCGGACCACATGAAACAGCACGACTTCTTCAAATCTGCTATGCCGGAAGGTTACGTTCAGGAACGTACCATCTTCTTCAAAGACGACGGTAACTACAAAACCCGTGCTGAAGTTAAATTCGAAGGTGACACCCTGGTTAACCGTATCGAACTGAAAGGTATCGACTTCAAAGAAGACGGTAACATCCTGGGTACAAACTGGAATACAACTACAACTCTCACAACGTTTACATCATGGCTGACAAACAGAAAAACGGTATCAAAGTTAACTTCAAAATCCGTCACAACATCGAAGACGGTTCTGTTCAGCTGGCTGACCACTACCAGCAGAACACCCCGATCGGTGACGGTCCGGTTCTGCTGCCGGACAACCACTACCTGTCTACCCAGTCTGCTCTGTCTAAAGACCCGAACGAAAAACGTGACCACATGGTTCTGCTGGAATTTGTTACCGCTGCTGGTATCACCCTGGGTATGGACGAACTGTACAAATAATAATACTAGAGCGCGGATCCGGTACCCCG 5CCGGGATCCTTCTTTAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGNTATACATATGCATCACCATCACCATCACGGCTCACTGGAAGTTCTGTTCCAGGGGCCCGGCTCAGTAAGCAAGGGCGAAGAGTTGTTTACCGGGGTTGTGCCGATCCTTGTCGAGCTTGACGGCGACGTAAACGGCCACAAGTTTAGCGTGTCCGGCGAAGGCGAAGGCGACGCAACGTACGGCAAGCTTACGCTTAAGTTTATCTGCACGACGGGCAAGCTTCCGGTGCCGTGGCCGACGCTTGTGACGACGCTTACGTACGGCGTGCAGTGCTTTAGCCGCTACCCAGATCACATGAAGCAACACGATTTCTTTAAGTCCGCAATGCCCGAAGGCTACGTCCAAGAGCGCACGATCTTCTTCAAAGACGACGGCAACTACAAGACGCGCGCAGAAGTGAAGTTTGAAGGCGATACGCTTGTGAACCGCATCGAGCTTAAGGGCATCGATTTCAAAGAAGACGGCAACATCCTTGGGCACAAGCTTGAGTACAACTACAACAGCCACAACGTCTATATCATGGCAGATAAGCAAAAGAACGGCATCAAGGTGAACTTTAAGATCCGCCACAACATCGAAGACGGCAGCGTGCAACTTGcAGATCACTACCAACAAAACACGCCGATCGGCGACGGCCCGGTGCTTCTTCCGGATAACCACTACCTTAGCACGCAATCCGCACTTAGCAAAGATCCGAACGAAAAGCGCGATCACATGGTCCTTCTTGAGTTTGTGACGGCAGCCGGCATCACGCTTGGCATGGACGAGCTTTACAAGTAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGCA GAGATCTCCG 6CCGGGATCCTTCTTTAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCATCACCATCACCATCACGGCTCACTGGAAGTTCTGTTCCAGGGGCCCGGCTCAGTAAGCAAGGGCGAAGAGTTGTTTACCGGGGTTGTGCCGATCCTTGTCGAGCTTGACGGCGACGTAAACGGCCACAAGTTTAGCGTGTCCGGCGAAGGCGAAGGCGACGCAACGTACGGCAAGCTTACGCTTAAGTTTATCTGCACGACGGGCAAGCTTCCGGTGCCGTGGCCGACGCTTGTGACGACGCTTACGTACGGCGTGCAGTGCTTTAGCCGCTACCCAGATCACATGAAGCAACACGATTTCTTTAAGTCCGCAATGCCCGAAGGCTACGTCCAAGAGCGCACGATCTTCTTCAAAGACGACGGCAACTACAAGACGCGCGCAGAAGTGAAGTTTGAAGGCGATACGCTTGTGAACCGCATCGAGCTTAAGGGCATCGATTTCAAAGAAGACGGCAACATCCTTGGGCACAAGCTTGAGTACAACTACAACAGCCACAACGTCTATATCATGGCAGATAAGCAAAAGAACGGCATCAAGGTGAACTTTAAGATCCGCCACAACATCGAAGACGGCAGCGTGCAACTTGCAGATCACTACCAACAAAACACGCCGATCGGCGACGGCCCGGTGCTTCTTCCGGATAACCACTACCTTAGCACGCAATCCGCACTTAGCAAAGATCCGAACGAAAAGCGCGATCACATGGTCCTTCTTGAGTTTGTGACGGCAGCCGGCATCACGCTTGGCATGGACGAGCTTTACAAGTAATAACTGCAGAGATCT CCG 7CCGGAATTCGGATCCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGCGTAAGGAGGTTTGGAATGCATCACCATCACCATCACGGCTCACTGGAAGTTCTGTTCCAGGGGCCCGGCTCAGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTCGAGCATTTACTGCTCGATTTACAGATGATTTTGAACGGCATTAATAATTACAAGAATCCTAAACTCACCCGCATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAAGAAGTGCTCAACTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTCGAACTAAAAGGCTCTGAAACAACATTCATGTGTGAATACGCTGACGAGACAGCAACCATTGTAGAATTTCTGAACCGTTGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGCGCGGATCCGG TACCCCG 8CCGGAATTCGGATCCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGCGTAAGGAGGTTTGGAATGCATCATCACCATCACCACGGCTCACTGGAAGTTCTGTTCCAGGGGCCCGGCTCAGCTCCGACCTCTTCTTCTACCAAAAAAACCCAGCTGCAGCTGGAACACCTGCTGCTGGACCTGCAGATGATCCTGAACGGTATCAACAACTACAAAAACCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCTACCGAACTGAAACACCTGCAGTGCCTGGAAGAAGAACTGAAACCGCTGGAAGAAGTTCTGAACCTGGCTCAGTCTAAAAACTTCCACCTGCGTCCGCGTGACCTGATCTCTAACATCAACGTTATCGTTCTGGAACTGAAAGGTTCTGAAACCACCTTCATGTGCGAATACGCTGACGAAACCGCTACCATCGTTGAATTTCTGAACCGTTGGATCACCTTCTGCCAGTCTATCATCTCTACCCTGACCTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGCGCGGATCCGGT ACCCCG

Linear double-stranded DNA was synthesized (GenScript, Inc.) with uniquerestriction sites at both the 5′ and 3′ ends (BamHI and BglII,respectively). To create the DNA mini-circle, the dsDNA was digestedwith both endonucleases to produce complementary sticky overhangs. Thisdigested DNA was ligated using T4 DNA ligase. Restriction digestion andligation steps were carried out either sequentially (e.g., in differenttubes) or simultaneously (e.g., in the same tube) using reactionmixtures containing 20 U BamH1, 10 U BglII, 400 U T4 ligase, 1 mM ATP,100 μg/mL bovine serum albumin (BSA), 100 mM NaCl, 10 mM MgCl₂, 50 mMTris-HCl, pH 7.5, and 10 mM dithiothreitol (DTT). All ligation products(DNA mini-circle) were subsequently treated with Exonuclease I andExonuclease III to digest any remaining linear DNA fragments. TheExonucleases were heat inactivated by incubating the ligation productsat 80° C. for 20 min. After heat-inactivation of the exonuclease, 5 μL(25 ng of DNA) of the completed ligation reaction was employed directlyfor isothermal RCA reactions using Phi29 DNA polymerase.

Amplification of the DNA Mini-Circle

The RCA of a DNA mini-circle template yields a high molecular weight,hyper-branched concatamer consisting essentially of tandem repeats of aminimalistic expression sequence. RCA reagents, including water,reaction buffer, primers, and phi29 enzyme were pre-cleaned prior to theaddition of ligated template and dNTPs to minimize off-targetamplification. In some embodiments, the primer-nucleotide solution(primer-nucleotide mix) containing an exonuclease-resistant primer andthe nucleotides (dNTPs) was decontaminated by incubating theprimer-nucleotide mix with a combination of exonuclease I, exonucleaseIII, and a single stranded DNA binding protein (SSB protein). The enzymemix containing a DNA polymerase was decontaminated by incubating with adivalent cation (e.g., Mg²⁺) optionally in presence of an exonuclease(if the DNA polymerase used included a non-proof-reading DNApolymerase). The amplification of the DNA mini-circle was performedusing such decontaminated enzyme mix and the primer-nucleotide mix. Forexample, the polymerase solution containing 200 ng of Phi29 DNApolymerase was incubated with 0.1 unit of exonuclease III in 5 μL of 50mM HEPES buffer (pH=8.0) containing 15 mM KCl, 20 mM MgCl₂, 0.01%Tween-20 and 1 mM TCEP. The incubation was performed either at 30° C.for about 60 min. or at 4° C. for 12 h. The decontaminated Phi29 DNApolymerase solution was transferred to an ice-bath and then was used inthe target RCA assay without prior inactivation of the exonuclease III.

The amplification of the DNA mini-circles was performed using randomhexamers or hexamer primers having the sequence +N+N(at N)(at N)(at N)*N(AT hexamers), where “N” represents a random nucleotide (i.e., N may beany of A, C, G, or T/U), “at N” represents a random mixture containing2-amino dA, 2-thio dT, normal G and normal C, a plus (+) sign precedinga letter designation denotes that the nucleotide designated by theletter is a locked nucleic acid (LNA) nucleotide, a star (*) signpreceding a letter denotes that the nucleotide designated by the letteris a phosphorothioate modified nucleotide. For all RCA reactions, thedNTP concentration was maintained below 1 mM (typically 400-800 μM) toavoid hydrogel formation of the amplified RCA product DNA, which canpotentially complicate the downstream process of transcription andtranslation from the RCA product.

DNA amplification reactions were performed by incubating thede-contaminated primer-nucleotide mix and the de-contaminated enzyme mixat 30° C. for about 400 min. with the DNA mini-circle template. Theamplification reaction mixture composed of 40 μM primer, 400 μM dNTPs(400 μM each of dATP, dCTP, dGTP, dTTP); 1 pg of DNA mini-circle, and200 ng phi29 DNA polymerase. The reaction mixture was incubated in 50 mMHEPES buffer (pH=8.0) containing 15 mM KCl, 20 mM MgCl₂, 0.01% (v/v)Tween-20, 1 mM TCEP. At the end of the incubation, the Phi29 DNApolymerase in the reaction mixture was inactivated by heating thereaction mixture at 65° C. for 10 minutes.

TABLE 3 and FIG. 2 summarize representative yields of the RCA productsfrom the DNA mini-circle prepared using SEQ ID No. 6, which was digestedwith BamHI and BglII and circularized by ligation. RCA reactions wereperformed under three different test conditions, (i) using randomhexamers and dNTPs. (ii) using AT-hexamer primers and dNTPs; and (iii)using AT hexamers and dNTPs mixed with thioated dATPs. RCA reactionswere performed in duplicate (where possible) using approximately 28 ngof exonuclease-treated ligation product (DNA mini-circles). All RCAreactions comprised 0.4 mM dNTP and 40 μM of primer (either randomhexamer or AT hexamer), except thioated dATP was added at a 1:40 ratiorelative to non-thioated dATP for one reaction (0.01 mM alpha-S-dATP).

RCA products were quantified using Quant-It™ Picogreen® dsDNA Assay Kit(ThermoFisher Inc.) from a total RCA reaction volume of 100 μL. Agarosegel electrophoresis of the restricted DNA products was also performed,and the intensity of the electrophoresis bands was compared to those ofstandards having known concentration of DNA. The yield of RCA productDNA by using AT hexamers, random hexamers, and AT hexamers and dNTPsmixed with thioated dATP (AT hexamer+α-S-dATP) using Quant-It PicoGreendsDNA Assay Kit are shown in Table 3. Only a single reaction for the RCAtest condition including AT hexamers and α-S-dATP was performed insteadof a duplicate reaction.

TABLE 3 Yield of RCA product DNA Reaction #1 Reaction #2 RCA DNA RCA DNARCA test condition yield (ng/μL) yield (ng/μL) Random hexamer 294.4323.3 AT hexamer 548.2 563.4 AT hexamer + α-S-dATP 590.8 —

As presented in Table 3 and FIG. 2, the yield of RCA product DNA isincreased when AT hexamer was used in comparison with the randomhexamers. As illustrated in FIG. 2, the yield of the RCA product wasapproximately 55 μg when AT hexamers were used for the RCA reaction,whereas the yield of RCA product was approximately 28 μg when randomhexamers were used for the RCA reaction. A similar high yield wasobserved when thioated bases were randomly incorporated into the RCAproduct after priming the amplification reaction with AT hexamers. Sincecoupled in vitro transcription and translation generally requires atleast 1 microgram of template DNA, the higher DNA yield using AT hexamerprimer in the RCA reactions enabled greater scale-up of subsequenttranscription and translation.

Various DNA amplification products (samples) were generated forcell-free expression comparison in the forthcoming examples, as listedin Table 4. Table 4 also illustrates the different sequence parameters(e.g., presence or absence of transcription termination sequence) and/ormodifications (e.g., presence or absence of thioated DNA, or codonusage) that are present in each template and the corresponding amplifiednucleic acids, wherein “cc” denotes a contextual codon preference and“ic” denotes an individual codon preference.

TABLE 4 Characteristics of different amplification products (samples)used in the forthcoming examples. Transcription Sample EncodingAmplification termination Template No. Gene method sequencesModification Sequence 20 EGFP RCA + cc Mini-circle of SEQ ID No. 1 22EGFP RCA − cc Mini-circle of SEQ ID No. 3 24 EGFP PCR + cc SEQ ID No. 126 EGFP PCR − cc SEQ ID No. 3 28 EGFP RCA + ic Mini-circle of SEQ ID No.2 30 EGFP RCA − ic Mini-circle of SEQ ID No. 4 32 EGFP PCR + ic SEQ IDNo. 2 34 EGFP PCR − ic SEQ ID No. 4 36 EGFP RCA + Thioated, Mini-circleof amplified SEQ ID No. 5 using AT hexamers; cc 38 EGFP RCA +non-thioated, Mini-circle of amplified SEQ ID No. 5 using AT hexamers;cc 40 EGFP RCA + non-thioated, Mini-circle of amplified SEQ ID No. 5using random hexamers; cc 42 EGFP PCR + Non- SEQ ID No. 5 thioated; cc44 EGFP RCA − Thioated, Mini-circle of amplified SEQ ID No. 6 using AThexamers; cc 46 EGFP RCA − Non- Mini-circle of thioated; SEQ ID No. 6amplified using AT hexamers; cc 48 EGFP PCR − Non- SEQ ID No. 6thioated; cc 50 IL-2 RCA + cc Mini-circle of SEQ ID No. 7 52 IL-2 PCR +cc SEQ ID No. 7 54 IL-2 RCA + ic Mini-circle of SEQ ID No. 8 56 IL-2PCR + ic SEQ ID No. 8 70 EGFP RCA + cc Mini-circle of SEQ ID No. 1 72EGFP RCA + cc Plasmid of SEQ ID No. 1 (in pUC19) 74 EGFP RCA − ccMini-circle of SEQ ID No. 3 76 EGFP RCA − cc Plasmid of SEQ ID No. 3 (inpUC19)

Example 2: Enhanced Protein Synthesis by Coupled In Vitro Transcriptionand Translation from RCA Product

Protein expression by coupled in vitro transcription and translation wasevaluated using the Expressway™ Mini Cell-Free Expression System(ThermoFisher Inc.), an E. coli-based coupled in vitro transcription andtranslation reaction that is suitable for linear and plasmid DNAtemplates. Per manufacturer specifications, the Expressway systemrequires a minimum concentration of DNA template of 148 ng/μL in orderto add 1 microgram of template DNA into a 50 μL reaction. Synthesis ofEGFP protein by coupled in vitro transcription and translation wascompared among PCR amplified DNA and RCA products prepared from DNA-minicircles derived from SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, or SEQ IDNo.4. All of these sequences contain a gene coding for EGFP, but differin several ways. For example, SEQ ID No.1 and SEQ ID No.2 comprise ClassI/II transcription terminator sequences derived from terminator T1 ofthe E. coli rrnB operon, whereas SEQ ID No.3 and SEQ ID No.4 lacktranscription termination sequences. Further, SEQ ID No.2 and SEQ IDNo.4 include EGFP open reading frames that are codon-optimized accordingto the JCat tool [Grote et al., Nucleic Acids Res. 2005 Jul. 1; 33 (WebServer issue: W526-531). JCat: a novel tool to adapt codon usage of atarget gene to its potential expression host], which maximizesindividual codon usage of a target gene to the codon preferences of anexpression host. SEQ ID No.1 and SEQ ID No.3 contain open reading framesfor EGFP that are contextually adapted based on the following process:starting from the natural coding sequence of EGFP, only specific siteswere re-coded to avoid cryptic start sites (ATG), cryptic ribosomalbinding sites (AGGA, GAGG, GGAG), class II termination sequences [(A, C,or T)ATCTGTT], ribosomal slippery sequences [NNNYYY, where Y=(A, T)],and ribosomal pause sites (AGG, GGA, GAG. GGG, GGT, GTG) upstream ofinternal ATG methionines.

DNA mini-circles containing sequences SEQ ID No.1, SEQ ID No.2, SEQ IDNo.3, and SEQ ID No.4 were prepared by ligation, and subsequentlyamplified by RCA with AT hexamers, as described in Example 1. Yield ofthe RCA product was quantified using Quant-It PicoGreen dsDNA Assay Kit(ThermoFisher Inc.). Approximately 1 microgram of RCA product derivedfrom DNA mini-circle template was applied directly to Expressway MiniCell-Free Expression reactions without any intermediate clean-up orpurification steps. Further, there was no additional processing of theRCA product in terms of restriction digestion and/or ligation before itsaddition to the Expressway Mini Cell-Free Expression reactions. Inseparate cell-free expression reactions, 1 microgram of PCR amplifiedDNA with SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, or SEQ ID No.4(synthesized by GenScript via overlap extension PCR) were also tested,along with no template controls (NTC). All cell-free expressionreactions were incubated at 30° C. for 6 hours with shaking (1200 rpm)in an Eppendorf ThermoMixer to synthesize the EGFP protein. Thesynthesized EGFP protein was allowed to fold into an active form byincubating it overnight at 4° C. prior to quantification by afluorescence-based assay. The fluorescence of folded EGFP was measuredfrom 10-fold dilutions of the expressed extract samples (in PBS) againsta purified EGFP reference curve (BioVision, Inc.) using a SpectraMax® M5Microplate Reader (Molecular Devices, LLC). Total EGFP yield from the invitro transcription and translation was calculated in units of μg/mL.

The EGFP protein yield from RCA product DNA prepared from SEQ ID No.1 orPCR amplified DNA prepared from SEQ ID No.3 are depicted in FIG. 3. FIG.3 illustrates that the EGFP expression was enhanced when RCA products 20and 22 were employed for in-vitro transcription-translation incomparison to in-vitro transcription-translation reactions using PCRamplified DNA 24 and 26. FIG. 3 also shows that RCA product 22 yieldedhigher EGFP expression as compared to PCR-amplified DNA 26 even thoughthe minimalistic expression sequence was devoid of any transcriptiontermination sequences.

The EGFP protein yield from an in-vitro transcription-translation of anRCA product prepared from SEQ ID No.2 or a PCR amplified DNA preparedfrom SEQ ID No.4 are depicted in FIG. 4. As depicted in FIG. 4, the EGFPyield was much higher when RCA products 28, 30 were used for cell-freeexpression when compared to the PCR-amplified DNA 32, 34. FIG. 4 alsoshows that RCA product 30 resulted higher yield of EGFP compared to thePCR amplified DNA 34 even when the minimalistic expression sequence wasdevoid of transcription termination sequences. The higher yield ofcell-free protein using an RCA product compared to PCR-amplified DNA wasunexpected. In general, the transcription termination signals arerequired for the stability and/release of mRNA and for effectivetranslation of the mRNA. For example, the protein expression from thePCR-amplified DNA 26 (FIG. 3) and 34 (FIG. 4) were substantially lowercompared to the PCR-amplified DNA 24 (FIG. 3) and 32 (FIG. 4)respectively. The minimalistic expression sequences of the PCR amplifiedDNA 24 and 32 have Class I/II transcription terminators (as shown inFIGS. 3 and 4) and the minimalistic expression sequences of the PCRamplified DNA 26 and 34 lack transcription terminators. Thus, this datashows that RCA product derived from minimalistic DNA mini-circlesimproves protein expression by generating tandem repeats of cistronicmRNA species, wherein each cistron contains the mRNA for the desiredtarget gene. The tandem repeats of the cistron may improve overall mRNAstability, particularly when transcription termination signals areabsent, and thus contribute to higher translational flux of the desiredprotein product.

Example 3: Thioation of RCA Product Improves Protein Production in aCoupled In Vitro Transcription-Translation System

Protein (EGFP) expression by coupled in vitro transcription andtranslation was evaluated using the Expressway Mini Cell-Free ExpressionSystem (ThermoFisher Inc) and PCR-amplified DNA or an RCA product. Forthese experiments, SEQ ID No. 5 and SEQ ID No. 6 were configured with astrong T7 gene10 translation enhancer sequence and T7 gene10 ribosomebinding sequence that increase translation efficiency. Additionally, SEQID No. 5 includes a Class I transcription terminator (derived from T7T-phi terminator sequence) which increases the termination efficiencydepending on reaction conditions and the upstream DNA sequence elements.The EGFP coding region was optimized contextually and was identicalbetween SEQ ID No. 5 and SEQ ID No. 6. DNA mini-circles were prepared byintramolecular ligation of each of the sequences, SEQ ID No.5 or SEQ IDNo. 6. The ligated DNA mini-circles were subsequently amplified by RCAwith random hexamers, AT hexamers, or AT hexamers in the presence orabsence of thioated dATPs, as described in Example 1. RCA yield wasquantified using Quant-It PicoGreen dsDNA Assay Kit (ThermoFisher). 0.5micrograms of RCA product DNA were applied directly into Expressway MiniCell-Free Expression reactions without any intermediate cleaning upsteps. In separate cell-free expression reactions, 0.5 micrograms of PCRamplified DNA of SEQ ID No. 5 or SEQ ID No. 6 (synthesized by GenScriptvia overlap extension PCR) were also tested, along with no templatecontrols (NTC). All cell-free expression reactions were incubated at 30°C. for 6 hours in an Eppendorf ThermoMixer (1200 rpm), and synthesizedEGFP protein was allowed to fold overnight at 4° C. prior tofluorescence quantitation. The fluorescence of folded EGFP was measuredfrom 100-fold dilutions of the extract samples (in PBS) against apurified EGFP reference curve (BioVision, Inc.) using a SpectraMax M5Microplate Reader (Molecular Devices, LLC). Total EGFP yield wascalculated in units of μg/mL.

EGFP protein yield from RCA products (including normal or thioated DNAderived from mini-circles prepared from SEQ ID No. 5) was comparedagainst the yield from PCR-amplified DNA (prepared from SEQ ID No. 5,without thioation). As depicted in FIG. 5, the EGFP protein expressionwas much higher from RCA product 36 that was partially substituted withthioated nucleotides, compared to PCR-amplified DNA 42, or RCA products38 and 40 that did not contain any thioated nucleotides.

Similarly, the expression yield of EGFP protein from SEQ ID No.6 wasdetermined from RCA products (with or without thioation) compared tonon-thioated PCR-amplified DNA. FIG. 6 shows the yield of EGFP wasrelatively higher for RCA product 44 that was partially substituted withthioated nucleotides, compared to the non-thioated RCA product 46 or anon-thioated PCR amplified template 48. For the PCR-amplified DNA 48,there was virtually no EGFP expression in the cell-free reaction (FIG.6). Similar to the previous Example, this data confirms thattranscription termination signals are required for effective translationof the mRNA, except when the minimalistic expression cassette isconverted into RCA product.

Example 4: RNA Polymerase Run-Off Influences Protein Production in an InVitro Transcription and Translation System Using an RCA Product in aContext-Dependent Manner

Expression of PCR-amplified DNA or RCA product in an in vitrotranscription and translation assay for human IL-2 was evaluated usingthe Expressway Mini Cell-Free Expression System (ThermoFisher Inc.). Forthese experiments, SEQ ID No. 7 and SEQ ID No. 8 (encoding IL-2 withouta signal peptide) were created with Class/II transcriptional terminatorsequences derived from terminator T1 of the E. coli rrnB operon. Theminimalistic expression sequences of SEQ. ID. No. 7 and SEQ ID No. 8were virtually identical except for codon usage within the IL-2 openreading frame. SEQ ID No.8 was codon-optimized according to the JCattool which maximized individual codon usage of a target gene to thecodon preferences of an expression host. SEQ ID No. 7 was contextuallyadapted based on the following process: starting from the natural codingsequence of human IL-2, only specific sites were re-coded to avoidcryptic start sites (ATG), cryptic ribosomal binding sites (AGGA, GAGG,GGAG), class II termination sequences [(A,C,T)ATCTGTT], ribosomalslippery sequences [NNNYYY, where Y=(A, T)], and ribosomal pause sites(AGG, GGA, GAG, GGG, GGT, GTG) upstream of internal ATG methionines. DNAmini-circles were prepared by intramolecular ligation of each of the DNAsequences, SEQ ID No. 7 and SEQ ID No.8. The mini-circles weresubsequently amplified by RCA with AT hexamers, as described inExample 1. Yield of the RCA product was quantified using Quant-ItPicoGreen dsDNA Assay Kit Assay Kit (ThermoFisher). 1 microgram of RCADNA was applied directly into Expressway Mini Cell-Free Expressionreactions without any intermediate clean-up step. In separate cell-freeexpression reactions, 1 microgram of PCR amplified DNA of SEQ ID No. 7and SEQ ID No. 8 (synthesized by GenScript via overlap extension PCR)were also tested, along with no template controls (NTC). Each cell-freeexpression reaction additionally contained 2 μL of FluoroTect™ GreenLYSin vitro Translation Label (Promega) to randomly label nascent lysineresidues (via anticodon UUU tRNA) with a fluorescent BODIPY-FL label.All cell-free expression reactions were incubated at 30° C. for 6 hoursin an Eppendorf ThermoMixer (1200 rpm) and then kept overnight at 4° C.prior to analysis. Approximately 2 μL of cell-free expression reactionmixture was separated by SDS-PAGE and all translation productscontaining BODIPY-FL were detected by in-gel fluorescence using aTyphoon Variable Mode Imager (GE Healthcare). Fluorescent IL-2 bands(˜16 kD) were digitally quantified using ImageJ software.

FIG. 7 demonstrates an SDS-PAGE gel for the IL-2 protein expressed usingcoupled in vitro transcription and translation. The expressed proteinswere collected from cell-free expression reactions and loaded toSDS-PAGE for analysis. The proteins were separately expressed from RCAproduct 50 prepared from SEQ ID No.7, RCA product 54 prepared from SEQID No.8, PCR-amplified DNA 52 prepared from SEQ ID No.7, andPCR-amplified DNA 56 prepared from SEQ ID No.8. As depicted in FIG. 7,aside from background BODIPY-FL tRNA signal that was also present in NTCreactions (lane 2), fluorescent signal from SEQ ID No. 7 waspredominately observed as ˜16 kD translated IL-2 protein (lanes 3-4). Incontrast, no IL-2 protein was observed for PCR-amplified DNA 56 (FIG. 7,lane 5). High-molecular weight proteins (26 kD-72 kD) were unexpectedlyexpressed using the RCA product 54 (SEQ ID No. 8), as shown in FIG. 7,lane 6. Lane 1 of FIG. 7 represents standard protein molecular weightmarker (M). The reason might be the presence of two di-lysine repeats inthe IL-2 sequence, which were re-coded into polyA tracts by the JCattool because the AAA codon is significantly preferred over AAG in E.coli. These di-lysine repeats were not substantially re-coded in SEQ IDNo. 7 by the contextual codon optimization process. AAAAAA tracts areoften known as ribosomal slippery sequences that can potentiallyframeshift the translated product (Yan S et al.; 2015, Ribosomeexcursions during mRNA translocation mediate broad branching offrameshift pathways; Cell. 160: 870-81) and exert additionaltranslational control through ribosomal stalling (Arthur et al., 2015,Translational control by lysine-encoding A-rich sequences; ScienceAdvances. Vol. 1: No. 6: pg 1-11; e1500154).

FIG. 8 demonstrates the relative yield of 16 kD IL-2 protein from RCAproduct 50 (prepared from SEQ ID No.7), RCA product 54 (prepared fromSEQ ID No.8), PCR-amplified DNA 52 (prepared from SEQ ID No.7), andPCR-amplified DNA 56 (prepared from SEQ ID No.8). FIG. 8 shows that theyield of IL-2 was increased when RCA product 50 was used for thecell-free expression compared to the PCR-amplified DNA 52. The datapresented in FIGS. 7 and 8 also show that tandem repeats of cistronicmRNAs are generated from RCA product despite the presence oftranscription termination sequences. The corresponding messages wereeffectively or ineffectively processed by ribosomes to affect cell-freeprotein yield, as long as the downstream cistrons in the message weredesigned appropriately.

Example 5: Protein Production in a Cell-Free Expression System Using anRCA Product Generated from a DNA Mini-Circle Compared to an RCA ProductGenerated from a Plasmid DNA

Expression of EGFP in a cell-free system was compared among RCA productsgenerated from DNA mini-circles consisting essentially of minimalisticexpression sequences using either SEQ ID No. 1 (having Class I/11transcriptional terminator sequence of the E. coli rrnB operon) or SEQID No. 3 (lacking a transcription terminator sequence). For theseexperiments, DNA mini-circles were prepared by intramolecular ligationof SEQ ID No. 1 or SEQ ID No. 3 as described in Example 1. For plasmidgeneration, SEQ ID No. 1 or SEQ ID No. 3 were ligated into the EcoRI andKpnI sites of pUC19, transfected into One Shot® TOP10 ChemicallyCompetent E. coli (Invitrogen #C4040), and selected for ampicillinresistance. Individual plasmid clones were purified from E. coli usingPureLink® HQ Mini Plasmid Purification Kit (Invitrogen #K2100-01) andverified by BamHI restriction digest. All DNA mini-circle and plasmidDNA templates were amplified by RCA with AT hexamers, as described inExample 1. RCA products were quantified using Quant-It PicoGreen dsDNAAssay Kit (ThermoFisher). 0.5 micrograms of RCA product DNA was applieddirectly into Expressway Mini Cell-Free Expression reactions without anyintermediate clean-up. Cell-free expression reactions (including notemplate controls) were incubated at 30° C. for 5 hours in an EppendorfThermoMixer (1200 rpm), and synthesized EGFP protein was allowed to foldkeeping at 4° C. for overnight prior to fluorescence quantitation. Thefluorescence of folded EGFP was measured from 10-fold dilutions of thelysate samples (in PBS) against a purified EGFP reference curve(BioVision, Inc) using a SpectraMax M5 Microplate Reader (MolecularDevices, LLC). Total EGFP yield was calculated in units of μg/mL.

Yield of the EGFP protein from cell-free expression reactions thatemployed RCA products amplified from DNA mini-circles (70, 74) and RCAproducts of plasmid DNA (72, 76) are depicted in FIG. 9. The resultsshow that the cell-free yield of EGFP is enhanced when RCA product isgenerated from DNA mini-circles rather than plasmid DNA. Further, EGFPcan be expressed even in the absence of transcription terminationsequences using RCA product derived from DNA mini-circles (74) comparedto RCA product derived from plasmid (76). This data shows that variousintervening sequences, such as origin of replication, antibioticselection sequence and extraneous sequences involved in clone screening,that are present in the RCA product generated from a plasmid DNA canimpair cell-free expression of the desired protein target. In contrast,beneficial and productive run-off can occur when the RCA concatemer isgenerated from a DNA mini-circle consisting essentially of aminimalistic expression sequence, even in the absence of a transcriptiontermination (which generally destabilizes mRNA transcripts).

The foregoing examples are illustrative of some features of theinvention, and are selected embodiments from a manifold of all possibleembodiments. The invention may be embodied in other specific formswithout departing from the spirit or essential characteristics thereof.While only certain features of the invention have been illustrated anddescribed herein, one skilled in the art, given the benefit of thisdisclosure, will be able to make modifications/changes to optimize theparameters. The foregoing embodiments are therefore to be considered inall respects as illustrative rather than limiting on the inventiondescribed herein. Where necessary, ranges have been supplied, and thoseranges are inclusive of all sub-ranges there between.

The invention claimed is:
 1. A method for in vitro transcriptioncomprising: generating a ribonucleic acid (RNA) in a cell-freetranscription reaction from a double-stranded rolling circleamplification (RCA) product comprising tandem repeats of a minimalisticdeoxyribonucleic acid (DNA) sequence of interest, wherein theminimalistic DNA sequence of interest comprises a promoter sequence andan RNA coding region, wherein the DNA sequence of interest is devoid ofa transcription termination sequence, and wherein the double-strandedRCA product is devoid of any extraneous sequences that are required forpropagation of a plasmid in a host cell.
 2. The method of claim 1,wherein the promoter sequence is located upstream of the RNA codingregion in the DNA sequence of interest.
 3. The method of claim 1,wherein the DNA sequence of interest further comprises an insulatorsequence.
 4. The method of claim 1, wherein the DNA sequence of interestfurther comprises a purification tag sequence.
 5. The method of claim 1,wherein the DNA sequence of interest is a codon-optimized sequence forenhancing RNA stability.
 6. The method of claim 1, wherein the generatedRNA comprises tandem repeats of cistronic RNA species, wherein eachcistronic RNA species comprises an RNA sequence coded by the DNAsequence of interest.
 7. The method of claim 1, wherein thedouble-stranded RCA product comprises a thioated nucleotide.
 8. Themethod of claim 1, wherein the cell-free transcription reaction isperformed using a prokaryotic cell extract, a eukaryotic cell extract,or a combination thereof.
 9. A method for in vitro transcription,comprising: generating a double-stranded rolling circle amplification(RCA) product via rolling circle amplification of a deoxyribonucleicacid (DNA) mini-circle comprising a minimalistic DNA sequence ofinterest; and generating a ribonucleic acid (RNA) in a cell-freetranscription reaction from the double-stranded RCA product, wherein theminimalistic DNA sequence of interest comprises a promoter sequence andan RNA coding region, wherein the DNA sequence of interest is devoid ofa transcription termination sequence, and wherein the DNA mini-circle isdevoid of any extraneous sequences that are required for propagation ofa plasmid in a host cell.
 10. The method of claim 9, wherein thedouble-stranded RCA product is used in the cell-free transcriptionreaction without any further processing.
 11. The method of claim 9,wherein the double-stranded RCA product comprises a thioated nucleotide.12. The method of claim 9, wherein the cell-free transcription reactionis performed using a prokaryotic cell extract, a eukaryotic cellextract, or a combination thereof.
 13. The method of claim 9, whereinthe DNA sequence of interest further comprises an insulator sequence.14. The method of claim 9, wherein the DNA sequence of interest furthercomprises a purification tag sequence.
 15. The method of claim 9,wherein the DNA sequence of interest is a codon-optimized sequence forenhancing RNA stability.
 16. The method of claim 9, wherein thegenerated RNA comprises tandem repeats of cistronic RNA species, whereineach cistronic RNA species comprises an RNA sequence coded by the DNAsequence of interest.
 17. The method of claim 9, wherein the rollingcircle amplification is performed using a final concentration ofdeoxyribonucleoside triphosphates (dNTPs) in a range of 10 μM to 10 mM.18. The method of claim 9, wherein the rolling circle amplification isperformed using a random primer mixture comprising a nucleotideanalogue, and wherein the nucleotide analogue is an inosine, a LockedNucleic Acid (LNA) nucleotide, a Peptide Nucleic Acid (PNA) nucleotide,a thioated nucleotide, 2-amino-deoxyadenosine, 2-thio-deoxythymidine, apolycation nucleotide, or a Zip Nucleic Acid (ZNA) polycation modifiednucleotide.
 19. The method of claim 18, wherein the random primermixture has a sequence +N+N(atN)(atN)(atN)*N.